K-ras gene mutation in colorectal adenomas and carcinomas from familial adenomatous polyposis patients

269

Surgical Oncology 1992 ; 1 : 269-274

K-ras gene mutation in colorectal adenomas and carcinomas from familial adenomatous polyposis patients I . S . BOUGHDADY*, A . R . KINSELLA`$, N . Y . HABOUBIt AND P . F . SCHOFIELDt `Paterson Institute for Cancer Research, Christie Hospital, Manchester, and i University Hospital of South Manchester, UK

Colorectal adenomas and carcinomas from familial adenomatous polyposis (FAP) patients were screened for the presence of K-ras gene mutations at codon 12 using an in vitro amplification step (polymerase chain reaction) followed by dot blot analysis using oligonucleotide probes specific for different mutations at codon 12 . We examined 28 colorectal adenomas and two colorectal carcinomas from 12 FAP patients and observed a mutation at codon 12 in seven adenomas and in both carcinomas . The frequency of K-ras gene mutations in colorectal tumours from FAP patients is similar to those in cases of sporadic adenomas and sporadic colorectal carcinomas indicating that the mechanisms involved in their development may be similar . Surgical Oncology 1992 ; 1 : 269-274 . Keywords : adenomas, carcinomas, codon 12, familial adenomatous polyposis, K-ras,

mutation .

INTRODUCTION Familial adenomatous polyposis (FAP) is an autosomally dominant disease, characterized by the presence or hundreds or thousands of colorectal adenomas . Surgically untreated individuals are at high risk of developing colorectal cancer [1] . Multiple genetic alterations occur during colorectal tumour development [2] . These changes include mutational activation of at least one oncogene, the ras gene, coupled with the mutational inactivation and/or loss of several tumour suppressor genes [2, 3] . We have reported a high incidence of K-ras gene mutations (35%) in sporadic colorectal neoplasms [4] . Farr et al . reported a low incidence of ras gene mutation (7%) in colonic adenomas from familial polyposis coli patients [5] and Sasaki et alL reported the frequency of K-ras gene mutations in colorectal carcinomas from FAP patients to be similar to that in sporadic colorectal carcinoma cases [6] . We now report on the type and incidence of K-ras Correspondence : Philip F . Schofield, MD, FRCS, Consultant Surgeon, Christie Hospital, Manchester, M20 9BX, UK .

gene mutations at codon 12 in colorectal tumours from patients with FAP .

MATERIAL AND METHODS The material for assay was taken from formalinized, paraffin-embedded histology blocks . Two carcinomas (three blocks analysed for each) and 28 adenomas (all less than 1 cm in diameter) were obtained from the pathology archives of South Manchester University Teaching Hospital . Sections were examined histologically to confirm the exact nature of the tissue analysed and to exclude blocks with marked tissue necrosis . DNA was extracted from 10 µm tissue sections by a modification of the method of Goelz [7] and as described by us [4] .

DNA amplification Selective amplification of the K-ras sequence which includes codon 12 was done using the polymerase chain reaction (PCR) technique (8, 9] . Taq polymerase (Perkin-Elmer Cetus) and two primers that flanked the region 3-68 of the K-ras gene were used



1. S. Boughdady et al .

270

to create an amplified 65 base pair (bp) K-ras gene

filters were exposed to Kodak XAR5 film for 48

fragment . The PCR cycle consisted of denaturation

hours at -80°C using an intensifying screen .

at 94°C (1 .5 min), primer annealing at 49°C (2 min) and polymerase mediated extension at 72°C (3 min) . After 30 cycles, and amplification of the correct 65

RESULTS

by sequence was confirmed by gel electrophoresis on 2% agarose .

Two colorectal carcinomas and 28 adenomas were obtained from 12 familial polyposis coli patients . Three autoradiograms illustrating the results of the

Oligonucleotide probes

hybridization reaction of amplified DNA extracted

The series of oligonucleotide probes used to detect normal and mutated sequences at codon 12 of the K-ras gene is listed in Table 1 . They were synthesized by ICI Pharmaceuticals (Cheshire, UK) and were end-labelled using 32P-ATP in the presence of T4 polynucleotide kinase .

from a group of tumour samples to three different oligonucleotide probes are shown in Fig . 1 . The results are summarized in Table 2 . K-ras gene mutations at codon 12 were observed in the two colorectal carcinomas examined in this group (samples 3, B, C, D and 6, C, D, E, Table 2) . Both carcinomas were clinicopathological stage B . Samples 3, B, C, D were from a well differentiated adenocarcinoma, while samples 6, C, D, E, were

Dot blot hybridization

from a moderately differentiated adenocarcinoma .

Amplified DNA fragments were dot blotted onto

In both carcinomas, mutations were GGT-GAT with

nylon membranes (hybond-N, Amersham Inter-

replacement of the normal glycine by aspartic acid .

national PLC, Bucks, UK) and hybridized in turn to

Mutations in the K-ras gene at codon 12 were

the radiolabelled probes listed above . The filters

also observed in seven of 28 adenomas (25%) .

were prehybridized for 16 h at 58°C in 6 x SSC,

Mutations resulted in replacement of the normal

0 .25% w :v low fat dried milk [10], and then hybridized for 3 h at 61°C in 10 ml of hybridization solution

glycine codon (GGT with valine (GTT) in three aden-

(6XSSC, 0 .25% w :v low fat dried milk 10% w :v dextran sulfate) with a pmol 32 P-labelled oligomer probe . The filters were then washed with 2 x SSC, 0 .25% w :v low fat dried milk, 0 .5% w :v SDS for 10

omas, with arginine (CGT) in two, with aspartic acid (GAT) in one, and with serine (GCT) in another adenoma . Four out of 22 tubular adenomas (18%) and three out of six tubulovillous adenomas (50%) contained

min at room temperature and at 56°C for 15 min .

mutant K-ras genes . According to the degree of

The filters were further washed with 1 x SSC, 0 .25%

dysplasia, two out of 15 polyps with mild dysplasia (13%) and five out of eleven polyps with moderate

w :v low fat dried milk, 0 .5% w :v SDS at 61°C for 10-15 min . This extensive washing improved the discrimination between a fully matched DNA hybrid and 1-bp-mismatched DNA hybrid considerably . The

dysplastic changes showed K-ras gene mutations at codon 12 . No mutations were detected in the two polyps with severe dysplasia .

Codon 12

Amino acid at Codon 12

Wild type sequence

5'

GTTGGAGCT GGT GGCGTAGG

Glycine

Oligonucleotides K-ras 12 WT

3'

CCAACCTCGA CCA CCGCATCC CAACCTCGA ACA CCGCATCC CAACCTCGA TCA CCGCATCC CAACCTCGA GCA CCGCATCC CAACCTCGA CAA CCGCATCC CAACCTCGA CIA CCTCATCC CAACCTCGA CGA CCTCATCC

Gly Cys Ser Arg Val Asp Ala

Table 1 . K-ras oligonucleotide series . Listed is the wild type sequence of the K-ras gene and the oligonucleotide series used in the hybridization experiments

K-ras gene mutation in FAP

Hybridization with Asparate-specific mutation probe (a)

Hybridization with Valine-specific mutation probe (b)

Figure 1 . Detection of K-ras gene mutation in colorectal tumours from FAP patients . The autoradiographs show samples with mutation as dark black spots . (a) Aspartate mutation . (b) Valine mutation . (c) Serine mutation . Numbers 64-70 correspond to patients 1-7 in Table 2 .

271

2 72

l. S. Boughdady

et al .

Hybridization with serine-specific mutation probe (c)

Figure 1 .-Continued. All the adenomas examined were less than 10 mm in size . The average size for the entire group of adenomas was 4 .6 mm, for those with mutations 5 .1 mm and for those without mutations 4 .3 mm . The above mentioned data could not be assessed statistically due to the small numbers included in the different groups. However, it is interesting to observe that identical mutations develop in the same patient . Patient 1 developed two mutations in two different polyps, both were due to replacement of glycine with valine (Fig . 1b, specimens 64B/C) . Similarly, patient 11 had the same type of mutation (arginine) in two different polyps .

DISCUSSION In this study K-ras gene mutations at codon 12 occurred in 25% of adenomatous polyps from FAP patients . This incidence is higher than that reported previously in comparable studies . Farr et al. [5], found ras gene mutations in only five out of 75 adenomas (7%) and Sasaki et al. [6] observed mutations at codon 12 of the K-ras gene in four out of 51 colorectal adenomas (8%) . We have shown that similar mutations occur in 35% of sporadic adenomatous polyps of the colorectum [4] . The

frequency of K-ras gene mutations in colorectal adenomas from FAP patients appears similar to that in sporadic adenomatous polyps suggesting that they may have similar mechanisms involved in their development . There was a higher incidence of mutations in adenomas with moderate dysplasia (45%) than those with mild dysplasia (13%) . Also, adenomas with mutations were larger in size than those without mutations . The types of mutations observed in adenomas from FAP patients were more variable than those in sporadic adenomas [4] . Mutations were detected that resulted in the replacement of the normal amino acid glycine with valine, arginine, aspartic acid and serine . The results from this study also suggest the presence of a certain predisposition to specific types of mutations of the K-ras gene in individual patients . One patient developed similar valine mutations in two different polyps, and another patient developed similar arginine mutations in two polyps . This differs from the data of Sasaki at alL which showed no predisposition to any specific type of mutation of the ras gene in individual patients . Farr et al. however, have noticed similar mutations in different polyps from the same patient . This tendency to develop a

273

K-ras gene mutation in FAP Table 2 . K-ras gene mutation in colorectal tumours from PFC patients

Sample

1A 1B 1C 1D 2A 2B 2C 3A 3B 3C 3D 4A 4B 5A 5B 6A 6B 6C 6D 6E 7A

Age/Sex

Specimen

Type

Dysplasia

Size (mm)

a .a . at codon 12

Mutation

31/F

Ad Ad Ad

T T T T T T T T -

3 3 5 3 4 5 3 5

gly gly val gly gly gly gly gly asp asp

gly val gly val

Ad Ad

++ + + + + + + + + + +

11/M

35/F

61/M 32/M

60/M

Ad Ad Ad Ca Ca Ca Ad Ad Ad Ad Ad Ad Ca Ca Ca Ad Ad Ad Ad Ad

T T T TV T T .V

11B 12A

30/M

Ad Ad

12B 12C

34/F

Ad Ad

T T

30/F

8B 9A 9B 10A 106

24/M 23/F 28/F

10C 11A

Ad Ad Ad Ad Ad

5 4 4 6 4

+ + +

5

T .V T .V T T T.V T .V T T T T T T

7B 8A

+++ + + + + +

+ + + + + + ++

7 6 4 5 3 4 8 5 6 5

+ + +

+ + ++ + + +

5 4 3 5

gly asp gly asp gly asp

gly gly gly ser gly asp asp asp asp val gly gly gly gly gly gly gly gly arg arg gly gly gly

gly asp gly gly gly gly gly

asp asp asp asp asp

gly arg glyarg

a .a .=amino acid . Ad=adenoma . Ca = carcinoma . T=tubular . T .V=tubulovillous . + = Mild . ++=moderate . +++= severe . val=valine .asp=aspartic . ser = serine . arg = arginine . gly= glycine .

specific type of mutation

in an individual patient

REFERENCES

could be the result of exposure to a single class of carcinogenic agent in the faecal stream . The two colorectal carcinomas from FAP patients examined, showed a glycine to aspartic acid mutation in both cases . Our other report [4] showed this to be the most common type of mutation to occur in cases of sporadic colorectal cancer . In conclusion, this study has shown that K-ras gene mutations occur in some colorectal tumours from familial adenomatous polyposis patients . These mutations are similar to those found in sporadic colorectal tumours and occur at the same fregency .

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